Quality control methods for the manufacture of polymer arrays

ABSTRACT

Described are quality control methods and devices for the reproducible manufacturing and integrity monitoring of polymers on electrochemical synthesis and detection chips. The devices and methods allow for simultaneous manufacturing and synthesis of polymers.

RELATED APPLICAITONS

This application is related to Attorney Docket # 07070-20052.00, andAttorney Docket # 07070-20054.00, filed herewith this application andthe disclosures of which are incorporated herein by reference.

FIELD OF INVENTION

The embodiments of the invention relate to quality control methods anddevices for the reproducible manufacturing and integrity monitoring ofpolymers on electrochemical synthesis and detection chips. The inventiontranscends several scientific disciplines such as polymer chemistry,biochemistry, molecular biology, medicine and medical diagnostics.

BACKGROUND

Molecular recognition (also called a binding event) is fundamental toevery cellular event: transcription, translation, signal transduction,viral and bacterial infection and immune response are all mediated byselective recognition events. Thus, developing a better understanding ofdetecting the binding events of molecules is of significant importance.

Typical methods for carrying out on-chip analyte detections include:optical tagging (fluorescence, visible, IR, Raman), radiometric (variousradioactive tag), and indirect electrochemical methods of detection(tagging with enzymes that generate charges that can be measured).

Polymer arrays are currently manufactured by three principal methods.One method employs printing/spotting technologies to deliver preformedpolymers onto attachment sites on the surface of a given substrate, mostoften ones composed of glass or quartz (e.g., see the methods ofAgilent.com). Quality controls for these attachment reactions that havebeen approached by this manufacturing method are necessarily indirect.For example, attachment can be measured by including fluorescentlytagged polymers into the reaction mixture that is spotted on the arraysso that binding can be detected by optical methods. Alternately,attached polymers can be detected by binding fluorescently tagged probesto the attached polymers

Another method for the manufacture of polymer arrays is to usephotolithographic methods to grow the polymers in situ. However, as withthe printing methods described above, the photolithographicmanufacturing processes are carried out on glass or quartz substrates.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of an exposed electrode array including large areadrive electrodes and micron or sub-micron scale sense and referenceelectrodes.

FIG. 2 is a schematic of exposed electrode array with attachmentchemistry and affinity probes attached to the sense and referenceelectrodes.

FIG. 3 is a schematic showing analyte (e.g. complementary sequence DNA)bound to affinity probe on a sense electrode.

FIG. 4 is a schematic of a device operating in differential detectionmode with key elements and electrical connections shown.

FIG. 5 is a schematic of device operating in absolute detection modewith key elements and electrical connections shown.

FIG. 6 is a circuit schematic of a detection device showing connectionof the sense and reference electrode capacitors, internal switches, etc.

FIG. 7 is a circuit schematic of the device showing connection of theaddressing circuits and attached amplifiers.

FIG. 8 is an image of a completed device prototype according to theinvention.

FIG. 9 is a graph showing how the device operates to detect analytebinding.

FIG. 10 is a schematic of a device in electrochemical synthesis ofpolymer affinity probe operation.

FIG. 11 is an alternative configuration of a device in electrochemicalsynthesis of polymer affinity probe operation.

FIG. 12 is an image of an electrode pattern in which concentric ringsare used to more effectively confine the acid/base field.

FIG. 13 is a graph of capacitance change as a function of the number ofreplicated targets.

FIG. 14 is graph of capacitance change as a function of time duringpolymer synthesis.

DETAILED DESCRIPTION

As used in the specification and claims, the singular forms “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “an array” may include a plurality ofarrays unless the context clearly dictates otherwise.

An “array” is an intentionally created collection of molecules which canbe prepared either synthetically or biosynthetically. The molecules inthe array can be identical or different from each other. The array canassume a variety of formats, e.g., libraries of soluble molecules;libraries of compounds tethered to resin beads, silica chips, or othersolid supports. The array could either be a macroarray or a microarray,depending on the size of the sample spots on the array. A macroarraygenerally contains sample spot sizes of about 300 microns or larger andcan be easily imaged by gel and blot scanners. A microarray wouldgenerally contain spot sizes of less than 300 microns.

“Solid support,” “support,” and “substrate” refer to a material or groupof materials having a rigid or semi-rigid surface or surfaces. In someaspects, at least one surface of the solid support will be substantiallyflat, although in some aspects it may be desirable to physicallyseparate synthesis regions for different molecules with, for example,wells, raised regions, pins, etched trenches, or the like. In certainaspects, the solid support(s) will take the form of beads, resins, gels,microspheres, or other geometric configurations.

The term “probe” or “probe molecule” refers to a molecule attached tothe substrate of the array, which is typically cDNA or pre-synthesizedpolynucleotide deposited on the array. Probes molecules are biomoleculescapable of undergoing binding or molecular recognition events withtarget molecules. (In some references, the terms “target” and “probe”are defined opposite to the definitions provided here.) Thepolynucleotide probes require only the sequence information of genes,and thereby can exploit the genome sequences of an organism. In cDNAarrays, there could be cross-hybridization due to sequence homologiesamong members of a gene family. Polynucleotide arrays can bespecifically designed to differentiate between highly homologous membersof a gene family as well as spliced forms of the same gene(exon-specific). Polynucleotide arrays of the embodiment of thisinvention could also be designed to allow detection of mutations andsingle nucleotide polymorphism.

The term “target” or “target molecule” refers to a small molecule,biomolecule, or nanomaterial such as but not necessarily limited to asmall molecule that is biologically active, nucleic acids and theirsequences, peptides and polypeptides, as well as nanostructure materialschemically modified with biomolecules or small molecules capable ofbinding to molecular probes such as chemically modified carbonnanotubes, carbon nanotube bundles, nanowires and nanoparticles. Thetarget molecule may be fluorescently labeled DNA or RNA.

The terms “die,” “polymer array chip,” “DNA array,” “array chip,” “DNAarray chip,” “bio-chip” or “chip” are used interchangeably and refer toa collection of a large number of probes arranged on a shared substratewhich could be a portion of a silicon wafer, a nylon strip or a glassslide.

The term “molecule” generally refers to a macromolecule or polymer asdescribed herein. However, arrays comprising single molecules, asopposed to macromolecules or polymers, are also within the scope of theembodiments of the invention.

“Predefined region” or “spot” or “pad” refers to a localized area on asolid support which is, was, or is intended to be used for formation ofa selected molecule and is otherwise referred to herein in thealternative as a “selected” region. The predefined region may have anyconvenient shape, e.g., circular, rectangular, elliptical, wedge-shaped,etc. For the sake of brevity herein, “predefined regions” are sometimesreferred to simply as “regions” or “spots.” In some embodiments, apredefined region and, therefore, the area upon which each distinctmolecule is synthesized is smaller than about 1 cm² or less than 1 mm²,and still more preferably less than 0.5 mm². In most preferredembodiments the regions have ann area less than about 10,000 μm² or,more preferably, less than 100 μm². Additionally, multiple copies of thepolymer will typically be synthesized within any preselected region. Thenumber of copies can be in the thousands to the millions. Morepreferably, a die of a wafer contains at least 400 spots in, forexample, an at least 20×20 matrix. Even more preferably, the diecontains at least 2048 spots in, for example, an at least 64×32 matrix,and still more preferably, the die contains at least 204,800 spots in,for example, an at least 640×320 array. A spot could contain anelectrode to generate an electrochemical reagent, a working electrode tosynthesize a polymer and a confinement electrode to confine thegenerated electrochemical reagent. The electrode to generate theelectrochemical reagent could be of any shape, including, for example,circular, flat disk shaped and hemisphere shaped.

An “electrode” is a body or a location at which an electrochemicalreaction occurs. The term “electrochemical” refers to an interaction orinterconversion of electric and chemical phenomena.

A “functionalized electrode” is an electrode of a microchip array havinga probe molecule that has a specific chemical affinity to a targetmolecule. An “unfunctionalized electrode” is an electrode of a microchiparray having no probe molecule or having a probe molecule that has nospecific chemical affinity to a target molecule.

The electrodes used in embodiments of the invention may be composed of,but are not limited to, metals such as iridium and/or platinum, andother metals, such as, palladium, gold, silver, copper, mercury, nickel,zinc, titanium, tungsten, aluminum, as well as alloys of various metals,and other conducting materials, such as, carbon, including glassycarbon, reticulated vitreous carbon, basal plane graphite, edge planegraphite and graphite. Doped oxides such as indium tin oxide, andsemiconductors such as silicon oxide and gallium arsenide are alsocontemplated. Additionally, the electrodes may be composed of conductingpolymers, metal doped polymers, conducting ceramics and conductingclays. Among the metals, platinum and palladium are especially preferredbecause of the advantageous properties associated with their ability toabsorb hydrogen, i.e., their ability to be “preloaded” with hydrogenbefore being used in the methods of the invention.

The electrodes may be connected to an electric source in any knownmanner. Preferred ways of connecting the electrodes to the electricsource include CMOS (complementary metal oxide semiconductor) switchingcircuitry, radio and microwave frequency addressable switches, lightaddressable switches, direct connection from an electrode to a bond padon the perimeter of a semiconductor chip, and combinations thereof. CMOSswitching circuitry involves the connection of each of the electrodes toa CMOS transistor switch. The switch could be accessed by sending anelectronic address signal down a common bus to SRAM (static randomaccess memory) circuitry associated with each electrode. When the switchis “on”, the electrode is connected to an electric source. Radio andmicrowave frequency addressable switches involve the electrodes beingswitched by a RF or microwave signal. This allows the switches to bethrown both with and/or without using switching logic. The switches canbe tuned to receive a particular frequency or modulation frequency andswitch without switching logic. Light addressable switches are switchedby light. In this method, the electrodes can also be switched with andwithout switching logic. The light signal can be spatially localized toafford switching without switching logic. This could be accomplished,for example, by scanning a laser beam over the electrode array; theelectrode being switched each time the laser illuminates it.

In some aspects, a predefined region can be achieved by physicallyseparating the regions beads, resins, gels, etc.) into wells, trays,etc.

A “protecting group” is a moiety which is bound to a molecule anddesigned to block one reactive site in a molecule, but may be spatiallyremoved upon selective exposure to an activator or a deprotectingreagent. Several examples of protecting groups are known in theliterature. The proper selection of protecting group (also known asprotective group) for a particular synthesis would be governed by theoverall methods employed in the synthesis. Activators include, forexample, electromagnetic radiation. ion beams, electric fields, magneticfields, electron beams, x-ray, and the like. A deprotecting reagentcould include, for example, an acid, a base or a free radical.Protective groups are materials that bind to a monomer, a linkermolecule or a pre-formed molecule to protect a reactive functionality onthe monomer, linker molecule or pre-formed molecule, which may beremoved upon selective exposure to an activator, such as anelectrochemically generated reagent. Protective groups that may be usedin accordance with an embodiment of the invention preferably include allacid and base labile protecting groups. For example, peptide aminegroups are preferably protected by t-butyloxycarbonyl (BOC) orbenzyloxycarbonyl (CBZ), both of which are acid labile, or by9-fluorenylmethoxycarbonyl (FMOC), which is base labile. Additionally,hydroxyl groups on phosphoramidites may be protected by dimethoxytrityl(DMT), which is acid labile. Exocyclic amine groups on nucleosides, inparticular on phosphoramidites, are preferably protected bydimethylformamidine on the adenosine and guanosine bases, and isobutyrylon the cytidine bases, both of which are base labile protecting groups.This protection strategy is known as fast oligonucleotide deprotection(FOD).

Any unreacted deprotected chemical functional groups may be capped atany point during a synthesis reaction to avoid or to prevent furtherbonding at such molecule. Capping groups “cap” deprotected functionalgroups by, for example, binding with the unreacted amino functions toform amides. Capping agents suitable for use in an embodiment of theinvention include: acetic anhydride, n-acetylimidizole, isopropenylformate, fluorescamine, 3-nitrophthalic anhydride and 3-sulfoproponicanhydride. Of these, acetic anhydride and n-acetylimidizole arepreferred.

Additional protecting groups that may be used in accordance with anembodiment of the invention include acid labile groups for protectingamino moieties: tertbutyloxycarbonyl, -tert-amyloxycarbonyl,adamantyloxycarbonyl, 1-methylcyclobutyloxycarbonyl,2-(p-biphenyl)propyl(2)oxycarbonyl,2-(p-phenylazophenylyl)propyl(2)oxycarbonyl,.alpha.,.alpha.-dimethyl-3,5-dimethyloxybenzyloxy-carbonyl,2-phenylpropyl(2)oxycarbonyl, 4-methyloxybenzyloxycarbonyl,benzyloxycarbonyl, furfuryloxycarbonyl, triphenylmethyl (trityl),p-toluenesulfenylaminocarbonyl, dimethylphosphinothioyl,diphenylphosphinothioyl, 2-benzoyl-1-methylvinyl, o-nitrophenylsulfenyl,and 1-naphthylidene; as base labile groups for protecting aminomoieties: 9-fluorenylmethyloxycarbonyl, methylsulfonylethyloxycarbonyl,and 5-benzisoazolylmethyleneoxycarbonyl; as groups for protecting aminomoieties that are labile when reduced: dithiasuccinoyl, p-toluenesulfonyl, and piperidino-oxycarbonyl; as groups for protecting aminomoieties that are labile when oxidized: (ethylthio)carbonyl; as groupsfor protecting amino moieties that are labile to miscellaneous reagents,the appropriate agent is listed in parenthesis after the group:phthaloyl (hydrazine), trifluoroacetyl (piperidine), and chloroacetyl(2- aminothiophenol); acid labile groups for protecting carboxylicacids: tert-butyl ester; acid labile groups for protecting hydroxylgroups: dimethyltrityl; and basic labile groups for protectingphosphotriester groups: cyanoethyl.

An “electrochemical reagent” refers to a chemical generated at aselected electrode by applying a sufficient electrical potential to theselected electrode and is capable of electrochemically removing aprotecting group from a chemical functional group. The chemical groupwould generally be attached to a molecule. Removal of a protectinggroup, or “deprotection,” in accordance with the invention, preferablyoccurs at a particular portion of a molecule when a chemical reagentgenerated by the electrode acts to deprotect or remove, for example, anacid or base labile protecting group from the molecule. Thiselectrochemical deprotection reaction may be direct, or may involve oneor more intermediate chemical reactions that are ultimately driven orcontrolled by the imposition of sufficient electrical potential at aselected electrode.

Electrochemical reagents that can be generated electrochemically at anelectrode fall into two broad classes: oxidants and reductants. Oxidantsthat can be generated electrochemically, for example, include iodine,iodate, periodic acid, hydrogen peroxide, hypochlorite, metavanadate,bromate, dichromate, cerium (IV), and permanganate ions. Reductants thatcan be generated electrochemically, for example, include chromium (II),ferrocyanide, thiols, thiosulfate, titanium (III), arsenic (III) andiron (II) ions. The miscellaneous reagents include bromine, chloride,protons and hydroxyl ions. Among the foregoing reagents, protons,hydroxyl, iodine, bromine, chlorine and thiol ions are preferred.

The generation of and electrochemical reagent of a desired type ofchemical species requires that the electric potential of the electrodethat generates the electrochemical reagent have a certain value, whichmay be achieved by specifying either the voltage or the current. Thereare two ways to achieve the desired potential at this electrode: eitherthe voltage may be specified at a desired value or the current can bedetermined such that it is sufficient to provide the desired voltage.The range between the minimum and maximum potential values could bedetermined by the type of electrochemical reagent chosen to begenerated.

An “activating group” refers to those groups which, when attached to aparticular chemical functional group or reactive site, render that sitemore reactive toward covalent bond formation with a second chemicalfunctional group or reactive site.

A “polymeric brush” ordinarily refers to polymer films comprising chainsof polymers that are attached to the surface of a substrate. Thepolymeric brush could be a functionalized polymer films which comprisefunctional groups such as hydroxyl, amino, carboxyl, thiol, amide,cyanate, thiocyanate, isocyanate and isothio cyanate groups, or acombination thereof, on the polymer chains at one or more predefinedregions. The polymeric brushes of the embodiment of the invention arecapable of attachment or stepwise synthesis of macromolecules thereon.

A “linker” molecule refers to any of those molecules described supra andpreferably should be about 4 to about 40 atoms long to providesufficient exposure. The linker molecules may be, for example, arylacetylene, ethylene glycol oligomers containing 2-10 monomer units,diamines, diacids, amino acids, among others, and combinations thereof.Alternatively, the linkers may be the same molecule type as that beingsynthesized (i.e., nascent polymers), such as polynucleotides,oligopeptides, or oligosaccharides.

The linker molecule or substrate itself and monomers used herein areprovided with a functional group to which is bound a protective group.Generally, the protective group is on the distal or terminal end of amolecule. Preferably, the protective group is on the distal or terminalend of the linker molecule opposite the substrate. The protective groupmay be either a negative protective group (i.e., the protective grouprenders the linker molecules less reactive with a monomer upon exposure)or a positive protective group (i.e., the protective group renders thelinker molecules more reactive with a monomer upon exposure). In thecase of negative protective groups, there could be an additional step ofreactivation. In some embodiments, this will be done by heating.

The polymer brush or the linker molecule may be provided with acleavable group at an intermediate position, which group can be cleavedwith an electrochemically generated reagent. This group is preferablycleaved with a reagent different from the reagent(s) used to remove theprotective groups. This enables removal of the various synthesizedpolymers or nucleic acid sequences following completion of thesynthesis. The cleavable group could be acetic anhydride,n-acetylimidizole, isopropenyl formate, fluorescamine, 3-nitrophthalicanhydride and 3-sulfoproponic anhydride. Of these, acetic anhydride andn-acetylimidizole are preferred.

The polymer brush or the linker molecule could be of sufficient lengthto permit polymers on a completed substrate to interact freely withbinding entities (monomers, for example) exposed to the substrate. Thepolymer brush or the linker molecule, when used, could preferably belong enough to provide sufficient exposure of the functional groups tothe binding entity. The linker molecules may include, for example, arylacetylene, ethylene glycol oligomers containing from 2 to 20 monomerunits, diamines, diacids, amino acids, and combinations thereof. Otherlinker molecules may be used in accordance with the differentembodiments of the present invention and will be recognized by thoseskilled in the art in light of this disclosure. In one embodiment,derivatives of the acid labile 4,4′-dimethyoxytrityl molecules with anexocyclic active ester can be used in accordance with an embodiment ofthe invention. More preferably,N-succinimidyl-4[bis-(4-methoxyphenyl)-chloromethyl]-benzoate is used asa cleavable linker molecule during DNA synthesis. Alternatively, othermanners of cleaving can be used over the entire array at the same time,such as chemical reagents, light or heat.

A “free radical initiator” or “initiator” is a compound that can providea free radical under certain conditions such as heat, light, or otherelectromagnetic radiation, which free radical can be transferred fromone monomer to another and thus propagate a chain of reactions throughwhich a polymer may be formed. Several free radical initiators are knownin the art, such as azo, nitroxide, and peroxide types, or thosecomprising multi-component systems.

“Living free radical polymerization” is defined as a livingpolymerization process wherein chain initiation and chain propagationoccur without significant chain termination reactions. Each initiatormolecule produces a growing monomer chain which continuously propagatesuntil all the available monomer has been reacted. Living free radicalpolymerization differs from conventional free radical polymerizationwhere chain initiation, chain propagation and chain terminationreactions occur simultaneously and polymerization continues until theinitiator is consumed. Living free radical polymerization facilitatescontrol of molecular weight and molecular weight distribution. Livingfree radical polymerization techniques, for example, involve reversibleend capping of growing chains during polymerization. One example is atomtransfer radical polymerization (ATRP).

A “radical generation site” is a site on an initiator wherein freeradicals are produced in response to heat or electromagnetic radiation.

A “polymerization terminator” is a compound that prevents a polymerchain from further polymerization. These compounds may also be known as“terminators,” or “capping agents” or “inhibitors.” Variouspolymerization terminators are known in the art. In one aspect, amonomer that has no free hydroxyl groups may act as a polymerizationterminator.

The term “capable of supporting polymer array synthesis” refers to anybody on which polymer array synthesis can be carried out, e.g., apolymeric brush that is functionalized with functional groups such ashydroxyl, amino, carboxyl etc. These functional groups permitmacromolecular synthesis by acting as “attachment points.”

The monomers in a given polymer or macromolecule can be identical to ordifferent from each other. A monomer can be a small or a large molecule,regardless of molecular weight. Furthermore, each of the monomers may beprotected members which are modified after synthesis.

“Monomer” as used herein refers to those monomers that are used to aform a polymer. However, the meaning of the monomer will be clear fromthe context in which it is used. The monomers for forming the polymersof the embodiments of the invention, e.g., a polymeric brush or a linkermolecule, have for example the general structure:

wherein R₁ is hydrogen or lower alkyl; R₂ and R₃ are independentlyhydrogen, or —Y—Z, wherein Y is lower alkyl, and Z is hydroxyl, amino,or C(O)—R, where R is hydrogen, lower alkoxy or aryloxy.

The term “alkyl” refers to those groups such as methyl, ethyl, propyl,butyl etc, which may be linear, branched or cyclic.

The term “alkoxy” refers to groups such as methoxy, ethoxy, propoxy,butoxy, etc., which may be linear, branched or cyclic.

The term “lower” as used in the context of lower alkyl or lower alkoxyrefers to groups having one to six carbons.

The term “aryl” refers to an aromatic hydrocarbon ring to which isattached an alkyl group. The term “aryloxy” refers to an aromatichydrocarbon ring to which is attached an alkoxy group. One of ordinaryskill in the art would readily understand these terms.

Other monomers for preparing macromolecules of the embodiments of theinvention are well-known in the art. For example, when the macromoleculeis a peptide, the monomers include, but are not restricted to, forexample, amino acids such as the L-amino acids, the D-amino acids, thesynthetic and/or natural amino acids. When the macromolecule is anucleic acid, or polynucleotide, the monomers include any nucleotide.When the macromolecule is a polysaccharide, the monomers can be anypentose, hexose, heptose, or their derivatives.

A “monomer addition cycle” is a cycle comprising the chemical reactionsnecessary to produce covalent attachment of a monomer to a nascentpolymer or linker, such as to elongate the polymer with the desiredchemical bond (e.g., 5′-3′ phosphodiester bond, peptide bond, etc.). Forexample, and not to limit the invention, the following steps typicallycomprise a monomer addition cycle in phosphoramidite-basedpolynucleotide synthesis: (1) deprotection, comprising removal of theDMT group from a 5′-protected nucleoside (which may be part of a nascentpolynucleotide) wherein the 5′-hydroxyl is blocked by covalentattachment of DMT, such deprotection is usually done with a suitabledeprotection reagent (e.g., a protic acid: trichloroacetic acid ordichloroacetic acid), and may include physical removal (e.g., washing,such as with acetonitrile) of the removed protecting group (e.g., thecleaved dimethyltrityl group), (2) coupling, comprising reacting aphosphoramidite nucleoside(s), often activated with tetrazole, with thedeprotected nucleoside, (3) optionally including capping, to truncateunreacted nucleosides from further participation in subsequent monomeraddition cycles, such as by reaction with acetic anhydride andN-methylimidazole to acetylate free 5′-hydroxyl groups, and (4)oxidation, such as by iodine in tetrahydrofuran/water/pyridine, toconvert the trivalent phosphite triester linkage to a pentavalentphosphite triester, which in turn can, be converted to a phosphodiestervia reaction with ammonium hydroxide. Thus, with respect tophosphoramidite synthesis of polynucleotides, the following reagents aretypically necessary for a complete monomer addition cycle:trichloroacetic acid or dichloroacetic acid, a phosphoramiditenucleoside, an oxidizing agent, such as iodine (e.g.,iodine/water/THF/pyridine), and optionally N-methylimidazole forcapping.

A “macromolecule” or “polymer” comprises two or more monomers covalentlyjoined. The monomers may be joined one at a time or in strings ofmultiple monomers, ordinarily known as “oligomers.” Thus, for example,one monomer and a string of five monomers may be joined to form amacromolecule or polymer of six monomers. Similarly, a string of fiftymonomers may be joined with a string of hundred monomers to form amacromolecule or polymer of one hundred and fifty monomers. The termpolymer as used herein includes, for example, both linear and cyclicpolymers of nucleic acids, polynucleotides, polynucleotides,polysaccharides, oligosaccharides, proteins, polypeptides, peptides,phospholipids and peptide nucleic acids (PNAs). The peptides includethose peptides having either α-, β-, or ω-amino acids. In addition,polymers include heteropolymers in which a known drug is covalentlybound to any of the above, polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, polyacetates, or other polymers which will beapparent upon review of this disclosure.

A “nanomaterial” as used herein refers to a structure, a device or asystem having a dimension at the atomic, molecular or macromolecularlevels, in the length scale of approximately 1-100 nanometer range.Preferably, a nanomaterial has properties and functions because of thesize and can be manipulated and controlled on the atomic level.

A “carbon nanotube” refers to a fullerene molecule having a cylindricalor toroidal shape. A “fullerene” refers to a form of carbon having alarge molecule consisting of an empty cage of sixty or more carbonatoms.

The term “nucleotide” includes deoxynucleotides and analogs thereof.These analogs are those molecules having some structural features incommon with a naturally occurring nucleotide such that when incorporatedinto a polynucleotide sequence, they allow hybridization with acomplementary polynucleotide in solution. Typically, these analogs arederived from naturally occurring nucleotides by replacing and/ormodifying the base, the ribose or the phosphodiester moiety. The changescan be tailor-made to stabilize or destabilize hybrid formation, or toenhance the specificity of hybridization with a complementarypolynucleotide sequence as desired, or to enhance stability of thepolynucleotide.

The term “polynucleotide” or “nucleic acid” as used herein refers to apolymeric form of nucleotides of any length, either ribonucleotides ordeoxyribonucleotides, that comprise purine and pyrimidine bases, orother natural, chemically or biochemically modified, non- natural, orderivatized nucleotide bases. Polynucleotides of the embodiments of theinvention include sequences of deoxyribopolynucleotide (DNA),ribopolynucleotide (RNA), or DNA copies of ribopolynucleotide (cDNA)which may be isolated from natural sources, recombinantly produced, orartificially synthesized. A further example of a polynucleotide of theembodiments of the invention may be polyamide polynucleotide (PNA). Thepolynucleotides and nucleic acids may exist as single-stranded ordouble-stranded. The backbone of the polynucleotide can comprise sugarsand phosphate groups, as may typically be found in RNA or DNA, ormodified or substituted sugar or phosphate groups. A polynucleotide maycomprise modified nucleotides, such as methylated nucleotides andnucleotide analogs. The sequence of nucleotides may be interrupted bynon-nucleotide components. The polymers made of nucleotides such asnucleic acids, polynucleotides and polynucleotides may also be referredto herein as “nucleotide polymers.

An “oligonucleotide” is a polynucleotide having 2 to 20 nucleotides.Phosphoramidites protected in this manner are known as FODphosphoramidites.

Analogs also include protected and/or modified monomers as areconventionally used in polynucleotide synthesis. As one of skill in theart is well aware, polynucleotide synthesis uses a variety ofbase-protected nucleoside derivatives in which one or more of thenitrogens of the purine and pyrimidine moiety are protected by groupssuch as dimethoxytrityl, benzyl, tert-butyl, isobutyl and the like.

For instance, structural groups are optionally added to the ribose orbase of a nucleoside for incorporation into a polynucleotide, such as amethyl, propyl or allyl group at the 2′-O position on the ribose, or afluoro group which substitutes for the 2′-O group, or a bromo group onthe ribonucleoside base. 2′-O-methyloligoribonucleotides (2′-O-MeORNs)have a higher affinity for complementary polynucleotides (especiallyRNA) than their unmodified counterparts. Alternatively, deazapurines anddeazapyrimidines in which one or more N atoms of the purine orpyrimidine heterocyclic ring are replaced by C atoms can also be used.

The phosphodiester linkage, or “sugar-phosphate backbone” of thepolynucleotide can also be substituted or modified, for instance withmethyl phosphonates, O-methyl phosphates or phosphororthioates. Anotherexample of a polynucleotide comprising such modified linkages forpurposes of this disclosure includes “peptide polynucleotides” in whicha polyamide backbone is attached to polynucleotide bases, or modifiedpolynucleotide bases. Peptide polynucleotides which comprise a polyamidebackbone and the bases found in naturally occurring nucleotides arecommercially available.

Nucleotides with modified bases can also be used in the embodiments ofthe invention. Some examples of base modifications include2-aminoadenine, 5-methylcytosine, 5-(propyn-1-yl)cytosine,5-(propyn-1-yl)uracil, 5-bromouracil, 5-bromocytosine,hydroxymethylcytosine, methyluracil, hydroxymethyluracil, anddihydroxypentyluracil which can be incorporated into polynucleotides inorder to modify binding affinity for complementary polynucleotides.

Groups can also be linked to various positions on the nucleoside sugarring or on the purine or pyrimidine rings which may stabilize the duplexby electrostatic interactions with the negatively charged phosphatebackbone, or through interactions in the major and minor groves. Forexample, adenosine and guanosine nucleotides can be substituted at theN² position with an imidazolyl propyl group, increasing duplexstability. Universal base analogues such as 3-nitropyrrole and5-nitroindole can also be included. A variety of modifiedpolynucleotides suitable for use in the embodiments of the invention aredescribed in the literature.

When the macromolecule of interest is a peptide, the amino acids can beany amino acids, including α, β, or ω-amino acids. When the amino acidsare α-amino acids, either the L-optical isomer or the D-optical isomermay be used. Additionally, unnatural amino acids, for example,β-alanine, phenylglycine and homoarginine are also contemplated by theembodiments of the invention. These amino acids are well-known in theart.

A “peptide” is a polymer in which the monomers are amino acids and whichare joined together through amide bonds and alternatively referred to asa polypeptide. In the context of this specification it should beappreciated that the amino acids may be the L-optical isomer or theD-optical isomer. Peptides are two or more amino acid monomers long, andoften more than 20 amino acid monomers long.

A “protein” is a long polymer of amino acids linked via peptide bondsand which may be composed of two or more polypeptide chains. Morespecifically, the term “protein” refers to a molecule composed of one ormore chains of amino acids in a specific order; for example, the orderas determined by the base sequence of nucleotides in the gene coding forthe protein. Proteins are essential for the structure, function, andregulation of the body's cells, tissues, and organs, and each proteinhas unique functions. Examples are hormones, enzymes, and antibodies.

The term “sequence” refers to the particular ordering of monomers withina macromolecule and it may be referred to herein as the sequence of themacromolecule.

The term “hybridization” refers to the process in which twosingle-stranded polynucleotides bind non-covalently to form a stabledouble-stranded polynucleotide; triple-stranded hybridization is alsotheoretically possible. The resulting (usually) double-strandedpolynucleotide is a “hybrid.” The proportion of the population ofpolynucleotides that forms stable hybrids is referred to herein as the“degree of hybridization.” For example, hybridization refers to theformation of hybrids between a probe polynucleotide (e.g., apolynucleotide of the invention which may include substitutions,deletion, and/or additions) and a specific target polynucleotide (e.g.,an analyte polynucleotide) wherein the probe preferentially hybridizesto the specific target polynucleotide and substantially does nothybridize to polynucleotides consisting of sequences which are notsubstantially complementary to the target polynucleotide. However, itwill be recognized by those of skill that the minimum length of apolynucleotide desired for specific hybridization to a targetpolynucleotide will depend on several factors: G/C content, positioningof mismatched bases (if any), degree of uniqueness of the sequence ascompared to the population of target polynucleotides, and chemicalnature of the polynucleotide (e.g., methylphosphonate backbone,phosphorothiolate, etc.), among others.

Methods for conducting polynucleotide hybridization assays have beenwell developed in the art. Hybridization assay procedures and conditionswill vary depending on the application and are selected in accordancewith the general binding methods known in the art.

It is appreciated that the ability of two single strandedpolynucleotides to hybridize will depend upon factors such as theirdegree of complementarity as well as the stringency of the hybridizationreaction conditions.

As used herein, “stringency” refers to the conditions of a hybridizationreaction that influence the degree to which polynucleotides hybridize.Stringent conditions can be selected that allow polynucleotide duplexesto be distinguished based on their degree of mismatch. High stringencyis correlated with a lower probability for the formation of a duplexcontaining mismatched bases. Thus, the higher the stringency, thegreater the probability that two single-stranded polynucleotides,capable of forming a mismatched duplex, will remain single-stranded.Conversely, at lower stringency, the probability of formation of amismatched duplex is increased.

The appropriate stringency that will allow selection of aperfectly-matched duplex, compared to a duplex containing one or moremismatches (or that will allow selection of a particular mismatchedduplex compared to a duplex with a higher degree of mismatch) isgenerally determined empirically. Means for adjusting the stringency ofa hybridization reaction are well-known to those of skill in the art.

A “ligand” is a molecule that is recognized by a particular receptor.Examples of ligands that can be investigated by this invention include,but are not restricted to, agonists and antagonists for cell membranereceptors, toxins and venoms, viral epitopes, hormones, hormonereceptors, peptides, enzymes, enzyme substrates, cofactors, drugs (e.g.opiates, steroids, etc.), lectins, sugars, polynucleotides, nucleicacids, oligosaccharides, proteins, and monoclonal antibodies.

A “receptor” is molecule that has an affinity for a given ligand.Receptors may-be naturally-occurring or manmade molecules. Also, theycan be employed in their unaltered state or as aggregates with otherspecies. Receptors may be attached, covalently or noncovalently, to abinding member, either directly or via a specific binding substance.Examples of receptors which can be employed by this invention include,but are not restricted to, antibodies, cell membrane receptors,monoclonal antibodies and antisera reactive with specific antigenicdeterminants (such as on viruses, cells or other materials), drugs,polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars,polysaccharides, cells, cellular membranes, and organelles. Receptorsare sometimes referred to in the art as anti-ligands. As the termreceptors is used herein, no difference in meaning is intended. A“Ligand Receptor Pair” is formed when two macromolecules have combinedthrough molecular recognition to form a complex. Other examples ofreceptors which can be investigated by this invention include but arenot restricted to:

a) Microorganism receptors: Determination of ligands which bind toreceptors, such as specific transport proteins or enzymes essential tosurvival of microorganisms, is useful in developing a new class ofantibiotics. Of particular value would be antibiotics againstopportunistic fungi, protozoa, and those bacteria resistant to theantibiotics in current use.

b) Enzymes: For instance, one type of receptor is the binding site ofenzymes such as the enzymes responsible for cleaving neurotransmitters;determination of ligands which bind to certain receptors to modulate theaction of the enzymes which cleave the different neurotransmitters isuseful in the development of drugs which can be used in the treatment ofdisorders of neurotransmission.

c) Antibodies: For instance, the invention may be useful ininvestigating the ligand-binding site on the antibody molecule whichcombines with the epitope of an antigen of interest; determining asequence that mimics an antigenic epitope may lead to the-development ofvaccines of which the immunogen is based on one or more of suchsequences or lead to the development of related diagnostic agents orcompounds useful in therapeutic treatments such as for auto-immunediseases (e.g., by blocking the binding of the “anti-self” antibodies).

d) Nucleic Acids: Sequences of nucleic acids may be synthesized toestablish DNA or RNA binding sequences.

e) Catalytic Polypeptides: Polymers, preferably polypeptides, which arecapable of promoting a chemical reaction involving the conversion of oneor more reactants to one or more products. Such polypeptides generallyinclude a binding site specific for at least one reactant or reactionintermediate and an active functionality proximate to the binding site,which functionality is capable of chemically modifying the boundreactant.

f) Hormone receptors: Examples of hormones receptors include, e.g., thereceptors for insulin and growth hormone. Determination of the ligandswhich bind with high affinity to a receptor is useful in the developmentof, for example, an oral replacement of the daily injections whichdiabetics take to relieve the symptoms of diabetes. Other examples arethe vasoconstrictive hormone receptors; determination of those ligandswhich bind to a receptor may lead to the development of drugs to controlblood pressure.

g) Opiate receptors: Determination of ligands which bind to the opiatereceptors in the brain is useful in the development of less-addictivereplacements for morphine and related drugs.

The term “complementary” refers to the topological compatibility ormatching together of interacting surfaces of a ligand molecule and itsreceptor. Thus, the receptor and its ligand can be described ascomplementary, and furthermore, the contact surface characteristics arecomplementary to each other.

A “scribe line” is typically an “inactive” area between the active diesthat provide area for separating the die (usually with a saw). Often,metrology and alignment features populate this area.

A “via” refers to a hole etched in the interlayer of a dielectric whichis then filled with an electrically conductive material, preferablytungsten, to provide vertical electrical connection between stacked upinterconnect metal lines that are capable of conducting electricity.

“Metal lines” within a die are interconnect lines. Metal interconnectlines do not typically cross the scribe line boundary to electricallyconnect two dies or, as in the some embodiments of this invention, amultitude of die to one or more wafer pads.

The term “oxidation” means losing electron to oxidize. The term“reduction” means gaining electrons to reduce. The term “redox reaction”refers to any chemical reaction which involves oxidation and reduction.

The term “wafer” means a semiconductor substrate. A wafer could befashioned into various sizes and shapes. It could be used as a substratefor a microchip. The substrate could be overlaid or embedded withcircuitry, for example, a pad, via, an interconnect or a scribe line.The circuitry of the wafer could also serve several purpose, forexample, as microprocessors, memory storage, and/or communicationcapabilities. The circuitry can be controlled by the microprocessor onthe wafer itself or controlled by a device external to the wafer.

The term “molecular binding event” means the occurrence of contactbetween a probe molecule and a target molecule. The devices fordetecting a molecular binding event according to the embodiments of thepresent invention are intended for use in a molecular recognition-basedassay for the analysis of a sample suspected of containing one or moretarget molecules or moieties such as specific nucleic acid sequences.The probe molecules of the array are provided for the purpose of bindingand detecting specific target molecules, e.g., nucleic acid sequences.The hybridization between the probe and target nucleic acid sequencesmay occur through the standard Watson-Crick hydrogen-bondinginteractions or other known specific binding interactions known in theart.

The term “polarization change” means a change in the amount ofcharge onan electrode produced by the deposition of a target molecule.

The term “differential amplifier” means a device that amplifies thedifference between two input signals (−) and (+). This amplifier is alsoreferred to as a differential-input single-ended output amplifier. It isa precision voltage difference amplifier, and could form the centralbasis of more sophisticated instrumentation amplifier circuits.

The term “field effect transistor” (FET) is a family of transistors thatrely on an electric field to control the conductivity of a “channel” ina semiconductor material. FETs, like all transistors, can be thought ofas voltage-controlled resistors. Most FETs are made using conventionalbulk semiconductor processing techniques, using the single-crystalsemiconductor wafer as the active region, or channel.

The term “CMOS” means complementary metal oxide semiconductor.

Embodiments include a method of synthesizing and monitoring thesynthesis of a polymer including activating an electrode to attach afirst polymer base to second polymer base on a surface of an electrode;detecting a capacitance change on the electrode surface; and activatingthe electrode to attach a third polymer base to the second polymer base.

In embodiments, the method may further include comparing the detectedcapacitance change to a pre-calibrated capacitance change. Thecomparison can be utilized to detect the density to at which polymersbases are being added on a given electrode, the extent to which apolymerization reaction has proceeded, or whether degradation/sideproduct reactions are occurring.

In some embodiments an integrating charge amplifier is connected to theelectrode and configured to detect capacitance changes at the electrodesurface. In some embodiments the polymer is a DNA oligonucleotide, apeptide, or an aptamer. Activating the electrode may include generatinga voltage on the electrode. The voltage may create an acidic or basicfield that deprotects a polymer base so that it binds on the electrodesurface.

In embodiments, the electrode is part of an array of electrodesfabricated on a single substrate.

Embodiments also include a polymer synthesis and monitoring deviceincluding a substrate; and an electrode on the surface of the substrate,wherein the device is configured to produce an activation cycle forbinding a polymer base on the electrode surface and a monitoring cyclefor measuring a capacitance change following an activation cycle.

Embodiments further include a method of quality control checking apolymer synthesis or polymer detection device including applying a testsolution to a device comprising an electrode; detecting a capacitancechange on the device; and comparing the detected capacitance change withan expected capacitance change.

In embodiments the test solution may include reagents that mimic theproperties of materials binding to, or reacting with, polymers on theelectrode surfaces. The comparison can be utilized to detect the densityto at which polymers bases are being added on a given electrode, theextent to which a polymerization reaction has proceeded, or whetherdegradation/side product reactions are occurring.

Embodiments also include a quality control kit including one or morequality control solutions, wherein the solutions are configured toproduce a specific capacitance change when applied to a polymersynthesis or detection device.

Described are methods for real-time detection of the binding ofanalytes. The devices do not require tagging, such as fluorescenttagging, in order to detect analytes. The methods are also potentiallymore sensitive than many methods of detection, which may reduce the needfor amplification if DNA is being detected. For example, experimentsshow charge detection with ˜5fF resolution. The devices and method alsopotentially offer a much broader dynamic range of operation, potentiallyas much as 7 orders of magnitude by precise control of manufacturing andlayout parameters (e.g. effective electrode area, amplifier gain,affinity probe uniformity, etc).

The same devices can also be used to synthesis polymers, particularlybio-polymers. The devices and methods offer unprecedented capabilitiesfor highly flexible polymer microarray synthesis at micron andsub-micron scale with reproducibility driven by CMOS manufacturing.

One embodiment is a method and apparatus for real-time detection of thebinding of analytes. More specifically, the methods and apparatusesdetect binding on an electrode surface or to an affinity probe attachedto an electrode surface on a CMOS device. The device may be made in astandard or modified CMOS process flow by one skilled in the art.

The device can be used to detect a wide variety of analyte bindings tothe electrode surface in many cases by attachment of an appropriateaffinity probe. Preferred bindings include, but are not limited to, DNAhybridization, peptide/protein binding, peptide phosphorylation,aptamer/protein binding and chemical bindings and adsorption.

Preferred detection devices include those that can be monolithicallyintegrated with the synthesis devices, for example field effect devices(direct and floating gate), impedance spectroscopy devices, integratingcharge amplifiers, and optical devices capable of being integrated withelectro-chemical synthesis devices. More preferred devices includeintegrating charge amplifiers.

One preferred embodiment of the integrating charge amplifier designincludes several components that can be implemented by one skilled inthe art in a traditional or modified CMOS process/fabrication process.The first element is a drive circuit that provides voltage pulses (canbe a variety of waveforms: square, sine, saw tooth, etc) to an exposeddrive electrode (i.e. accessible to the introduction of analyte, fluid,or gas). The second is an integrating charge amplifier that has oneinput as an exposed sensing electrode and the other as an exposed orunexposed reference electrode.

The exposed reference electrode allows for common mode noise rejectionby inputting to one input of the amplifier a signal representing thesame environmental conditions (pH, temperature, ion concentration,non-binding analytes, etc). Alternatively, the reference capacitor canbe exposed to air or covered to establish an absolute reference. Anexample, of such an embodiment is described below with respect to FIG.5.

To the sense electrodes, affinity probes can be attached throughchemical methods. The reference electrode may or may not have a similaror different affinity probe attached and each sensing electrode can havea different affinity probe attached to detect a variety of analytes(e.g. multiple genes in a sample). The integrated charge value can beconverted to a voltage through a two stage amplifier. An internal (notexposed, monolithic NMOS or Metal-Insulator-Metal) capacitor may beconnected to the amplifier via an internal switch to be used as areference capacitor. These elements provide the driving, sensing, andreferencing capability of the device. FIG. 1 shows these elements ineach reference configuration. In FIG. 1 shows an exposed electrode arrayincluding large area drive electrodes and micron or sub-micron scalesense and reference electrodes. The array can be of any densitymanufacturable by standard semiconductor fabrication techniques.

FIG. 2 shows a representation of sense and reference electrodes coatedwith chemistries of attachment and attached DNA probes (scales are notrelative, ˜100,000 single strand DNA oligonucleotides fit in a 1 um2area. Also, probe length may vary, with lengths between 24-mer and60-mer preferred).

FIG. 3 shows a representation of attached single strand DNA probeunhybridized and a double strand DNA representing a probe that has founda complementary match from the sample solution and hybridized.

A device including one or more integrating charge amplifiers ispreferably configured to measure the integrated charge and effectivecapacitance at the analyte-electrode interface. A change in integratedcharge or effective capacitance can then be used to ascertain whetherthe analytes have bound at the electrode surface or to the affinityprobe attached to the electrode surface. This configuration need not bespecific to any analyte but preferably can be applied to all analytes.

When measuring the effective capacitance, preferably the analyte isprovided in a conductive solution that provides a conductive pathbetween the driving and integrating electrodes of the amplifier, forexample a conductive solution, such as liquid aqueous solution, or aconductive gel. A preferred method of using the device including one ormore integrating charge amplifiers includes providing a voltage pulsethrough the drive electrode to the conductive matrix including theanalyte. This pulse can be applied to the matrix with respect to anintegrating electrode and the charge can be accumulated on theintegrating electrode over a fixed time.

The measured capacitance is established by the fixed sensing electrode,the dielectric formed by the attachment chemistry, attached probe, andbound analyte (if a match is found), and a virtual parallel plate formedabove the sense electrodes by the charge/ion distribution in the matrix.The measured capacitance is a function of the electrode area, thedielectric constant, and the distance of the virtual plate from thesensing electrode. Analytes binding to the electrode or attachedaffinity probe will change the dielectric constant and/or distancebetween the virtual plate and sense electrode, thereby changing theeffective capacitance and accumulated charge on the sense electrode whena voltage is applied. The area and distance to the drive electrode arenot material since the conductive matrix carries the voltage to thevirtual plate. Any capacitance contributed by the drive electrode is inseries with the measured capacitance and is small owing to the largeelectrode area.

To compensate for any noise at the time of the test (due to 1/f noise,thermal noise, etc.) a calibrating reference pulse is preferably appliedto an internal test capacitor to normalize the response of the amplifierduring each measurement cycle (this is different from the referenceelectrode). The output of this amplifier can then be digitized andpost-processed. Post-processing is an algorithm that is applied throughsoftware to remove random noise, slopes, etc. The parameters can bedetermined experimentally by characterizing the various contributingparameters: e.g electrode size, drive voltage, environmental conditionssuch as temperature, analyte binding concentration, etc.

Preferably, individually addressable sensing electrode arrays of variouseffective areas are created to increase the detection range of theamplifier to various concentrations of target in the solution. A largearray of driving electrodes can be created to allow close coupling ofdriving voltage to the solution. Since the drive electrode capacitanceis in series with the sense electrode interface+probe/target through thesolution, preferably the driving electrode area is larger than sensingelectrode area to reduce parasitic effects. A system comprised of alarge capacitor in series with a small capacitor is dominated by thesmall capacitor. In this case: 1/Cseries=1/Cintegrated+1/Cdrive->1/Cintegrated as Cdrive gets large.

An array of integrating amplifiers and respective electrode arrays arepreferably fabricated on the same substrate. (also synthesis anddetection drive circuits, may also include logic for switching, latches,memory, IO devices, and other device able to be integrated in silicon)

For improved sensitivity, preferably a device that includes one or morereference electrodes is used for differential measurements. Referenceand sensing electrode routing paths can be matched in a layout patternto reduce parasitic coupling.

Preferably, the measurement of the change in capacitance at the sensingelectrode is accomplished in one of two manners. First, the change canbe detected with respect to the exposed reference capacitor. In thisembodiment, the reference electrode is exposed to the same solution asthe sensing electrode. Preferably, a probe that is designed to havesimilar electrical characteristics as the affinity probed but not tobind to a target in the solution in attached to the reference electrode.A change in integrated charge is measured as binding occurs on thesensing electrode (or affinity probe attached thereon) whose electricalcharacteristics change, but not on the reference electrode whoseelectrical characteristic remain the same. Second, two measurements ofthe same electrode, before and after the analyte binds, can be comparedto establish the change in integrated charge resulting from binding. Inthis case, the same electrode at a previous time provides the reference.FIG. 4 shows an example of a device configured to operate in thisfashion. In FIG. 4 the dotted lines show resistance and capacitive pathsestablished by the conductive matrix and insulating affinityprobe/analyte layer on the electrode. This schematic shows the deviceoperation in differential detection mode, in which both reference andsense electrode have attached affinity probes (of different affinity) toreject common mode noise contributed by the matrix and other parasiticsand noise sources.

In an alternative configuration, the reference electrode can beconfigured so that the sensing electrode takes direct capacitancemeasurements (non-differential). In this configuration, the referenceelectrode can be covered with a small dielectric substance such as epoxyor the device passivation or left exposed to air. The signal from theelectrode can then be compared to an open circuit which establishes anabsolute reference for measurement but may be more susceptible to noise.FIG. 5 shows an example of a device in an absolute detection mode, inwhich the reference is an unexposed (or exposed to a fix environmentsuch as air) fixed capacitor.

This device preferably provides real-time detection capability. In moretraditional approaches, all of the sample analytes are tagged with anoptical label (Cy dye, FITC, etc.). To determine what has bound to thesurface, the sample must be washed off after a time to remove unboundanalytes and their associated labels. This wash stops all kinetics ofthe analytes and electrode/probes. In this invention, analytes are nottagged and detection is electrical, so washing is not needed to removeunwanted signal. Therefore real-time monitoring of binding kinetics canbe accomplished by providing dynamic measurement at the solid-solutioninterface by applying a pulse and integrating the response at varioustimes during the development of hybridization. In other words, since nowash is required to remove the unbound but labeled analytes, theinvention can operate without the experiment being halted with the wash.

Preferably, the device has a digital mode capability. For example, a 1bit latch can be provided on the chip device. The response of theamplifier to a drive or reference pulse can then be stored in thislatch. For instance, the response can be above or below the response ofthe reference electrode. By varying the value of the drive or referencepulses and capturing the responses to these pulses more than 1 bitresolution can be obtained. Parallel operation of a large number ofsensing nodes and amplifiers can be done this way. Alternatively, ananalog signal can be read out directly or digitized through an internalor external analog to digital converter.

FIG. 6 is a circuit schematic of a detection device showing connectionof the sense and reference electrode capacitors, internal switches, etc.

FIG. 7 is a circuit schematic of the device showing connection of theaddressing circuits and attached amplifiers.

FIG. 8 is an image of a completed device prototype according to theinvention.

FIG. 9 is a graph showing how the device operates to detect anyltyebinding. In FIG. 9 device measurement of integrated charge on twoelectrode: the first (red) with attached affinity probe only and thesecond (green) with analyte bound to attached affinity probe. In thisexperiment, the affinity probe and analyte are single strandoligonucleotides.

Another embodiment is a method and device for electrochemicallysynthesize polymers. The electrodes may be the same electrodes used todetect analyte bindings with circuitry to connect them to either drivecircuitry or the integrating charge amplifiers.

Preferred polymers that are manufactured include, but are not limitedto, DNA oligonucleotides, peptides, and aptamers.

The electrodes may be made from a variety of materials utilized instandard CMOS fabrication. Preferred electrode materials include Ti,TiN, and Ta. Alternatively, materials that are compatible with a CMOSfabrication process but not traditionally used, such as Au and Pt, canbe employed in this invention.

During polymer synthesis, the electrodes are used to control the acidicor basic region directly above them. For example, micron and/orsub-micron exposed surface area, confinement electrodes of oppositepolarity or floating separate attachment electrodes that may be groundedor floating. Having these drive voltage options allows the user tooptimize the chemistry for the synthesis preferred. For example, thedrive electrode can source the voltage that creates the acid field, thefloating electrode can be placed so that the chemistry is optimal forsynthesis, and the electrode of opposite polarity can quench the acid orbase to confine the reaction to the desired area. One skilled in the artwill understand how to appropriately drive these electrodes to maximizethe electrochemical synthesis process. Preferably, the electrode onwhich the polymer is synthesized is also the electrode connected viacircuitry to the integrating charge amplifier.

Preferably, large arrays of electrodes are arranged on a substrate. Theelectrodes are preferably, in various arrangements and sizes on the samesubstrate to enable a diversity of polymers and multiplexing ofindividual electrodes to create polymers of desired sizes and shapes.

Preferably, one or more distinct voltage pulses can be applied to theeach of the electrodes. This allows creation of an active or group ofactive electrodes surrounded by an array of confinement electrodeswithout addition of any other special function electrodes. In otherwords, multiple electrodes can be similarly driven to create largerregions of acid or base field. Likewise, multiple electrodes can be usedto form a common attachment or quenching region.

FIG. 10 is an example of a device in electrochemical synthesis ofpolymer affinity probe operation. The acid/base field generated by theactivated electrode deprotects an existing attachment chemistry orpolymer base allowing subsequent base attachment. Note the acid/basefield is free to diffuse across the surface of the device when otherelectrodes are inactive. Also note that the exposed electrodes can bethe same as those used in the detection operation.

FIG. 11 is an alternative configuration of a device in electrochemicalsynthesis of polymer affinity probe operation. The acid field generatedby the activated electrode deprotects an existing attachment chemistryor polymer base allowing subsequent base attachment. Note the acid fieldconfined in this mode of operation when surrounding electrodes areactivated to the opposite polarity (or otherwise driven appropriately toquench the acid or base field). Again note that the exposed electrodescan be the same as those used in the detection operation.

In another embodiment, electrode can be used that have arbitrary sizeand shape to define the chemistry. For example, a confinement electrodecan be a ring of metal around the drive and/or floating electrode. FIG.12 is an image of a synthesis electrode pattern in which concentricrings are used to more effectively confine the acid/base field. Againnote that the exposed electrodes can be the same as those used in thedetection operation. The electrode size and shape are immaterial.

Preferably, a set of two latches are provided at each active electrodesite to allow the electrode to exist in three states driven by voltage1, driven by voltage 2, or floated during the synthesis cycle.

The voltage sources for the electrodes on the device can be internallymultiplexed from external sources through digital control, andpreferably can be applied in parallel to a large array of electrodes.

In application, voltages are applied to a programmed selection ofelectrodes as a solution of polymer base is flowed over the electrodes.The applied voltage creates an acidic or basic field that deprotects thepolymer base so that it attaches to the electrode surface, surfacechemistry, or previous base. By flowing different bases over theelectrodes with the desired programmed electrode activation, the polymersequence is generated at each electrode.

Yet another configuration is a method and device to synthesize polymersand detect binding to the polymer on a common integrated device surface.The device may be the same device used to detect analyte bindings andsynthesize polymers previously described. The device is capable ofcarrying out on-chip electrochemical polymerization reactions(functionalization) and also on-chip label-free analyte detection.

Polymers that can be synthesized and detected utilizing the devices andmethods include, but are not limited to, DNA oligonucleotides, peptides,and aptamers. Bindings include, but are not limited to, DNAhybridization, peptide/protein binding, peptide phosphorylation, andaptamer/protein binding. Detection devices include those that can beintegrated with the synthesis devices, for example field effect devices(direct and floating gate), impedance spectroscopy devices, integratingcharge amplifiers, and optical devices, capable of being integrated withelectro-chemical synthesis devices. Preferred devices include devicesthat utilize integrated charge amplifiers.

In one preferred configuration, the same CMOS circuitry that enables thevoltage outputs of the electrochemical synthesis reactions above aredesigned to be easily re-programmed for electrical detection. Inparticular, the sensing AND synthesis electrodes can be individuallyaddressed, and any electrode can behave as detection, driving, activesynthesis or shielding electrode.

Preferably, the device includes one or more integrating chargeamplifiers and one or more electrodes capable of being driven to a givenvoltage in a pulsed manner, or in any arbitrary shape. Preferably, largearrays of electrodes of various sizes are arranged on the samesubstrate.

Preferably, one or more voltage patterns can be applied to the each ofthe electrodes. This allows creation of an active or group of activeelectrodes surrounded by an array of shielding electrodes withoutaddition of any other special function electrodes

Preferably, a set of two latches are provided at each site to allow theelectrode to exist in the three states: driven by voltage 1, driven byvoltage 2, or floated during the synthesis cycle.

Preferably, the voltage sources are internally multiplexed from externalsources through digital control, and can be applied in parallel to alarge array of electrodes. By modulating voltages at electrodes on acase-by-case basis, synthesis chemistries can by tailored to theparticular requirements of each electrode.

The same CMOS circuitry and electrodes used for voltage outputs of theelectrochemical synthesis reactions can also be used to detect bindings.Integrating charge amplifiers are preferably utilized to measure thechanges in charge and effective capacitance at the analyte-electrodeinterface.

Preferably, a pulse of voltage is applied to the solution with respectto an integrating electrode and the charge is accumulated over a fixedtime. A calibrating reference pulse can be applied to the solutionthrough an internal test capacitor to normalize the response of theamplifier during each measurement cycle.

Preferably, a two stage integrating charge amplifier to convert thecharge to a voltage.

Logic circuits can be used to digitize and post-process the output ofthe two stage integrating amplifier. Post-processing removes randomnoise, slopes, etc. These circuits can be on the same chip or off chip.

Individually addressable sensing electrode arrays of various effectiveareas can be used to increase detection range. Preferably, a large arrayof driving electrodes is created to allow close coupling of drivingvoltage to the solution. In addition, preferably the driving electrodearea is larger than sensing electrode area to reduce parasitic effects.

In application, voltages are applied to a programmed selection ofelectrodes as a solution of polymer base is flowed over the electrodes.The applied voltage creates an acidic or basic field that deprotects thepolymer base so that it attaches to the electrode surface, surfacechemistry, or previous base. By flowing different bases over theelectrodes with the desired programmed electrode activation, the polymersequence is generated at each electrode.

A sample solution can be applied to the electrode array with polymerssequences attached. Where the analytes in the sample have affinity withthe attached polymer, they bind.

The detection circuits can used with the same electrodes as describedabove to detect the change in capacitance of the electrode interface. Inthis manner, an integrated synthesis/detection device can use a commonset of electrodes to synthesize polymers and detect binding. This methodoffers unprecedented capabilities for merging highly flexible polymer

Real Time Polymerase Chain Reaction (PCR) Monitoring

Progress of DNA amplification during a Polymerase Chain Reaction (PCR)can be monitored in “real time” by measuring capacitance change at eachfeature location. Reaction rates can be measured continuously, ordetermined at a fixed time point during the exponential amplificationphase. A computer can be used to measure the rate of capacitance changeof all simultaneous experimental PCR reactions occurring on thedetection chip. Unlike ordinary preparative PCR, RT-PCR allows thesuccess of multiple PCR reaction to be determined automatically afteronly a few cycles, without separate analysis of each reaction, andavoids the problem of “false negatives”.

PCR Assay Protocol

Typical PCR protocols can be followed. The number of copies of total RNAused in the reaction are preferably enough to give a signal by 25-30cycles. The optimal concentrations of the reagents are as follows: i.Magnesium chloride concentration should be between 4 and 7 mM. ii.Concentrations of dNTPs should be balanced with the exception of dUTP(if used). iv. The optimal probe concentration is 50-200 nM, and theprimer concentration is 100-900 nM. AmpErase uracil-N-glycosylase (UNG)is added in the reaction to prevent the re-amplification of carry-overPCR products by removing any uracil incorporated into amplicons. It isnecessary to include at least three No Amplification Controls (NAC, aminus-reverse transcriptase control) as well as three No TemplateControls (NTC, a minus sample control) in each reaction plate (toachieve a 99.7% confidence level in the definition of +/−thresholds forthe target amplification, six replicates of NTCs must be run). NAC is amock reverse transcription containing all the RT-PCR reagents, exceptthe reverse transcriptase; NTC includes all of the RT-PCR reagentsexcept the RNA template.

Electrochemical Biochip Design

To configure the chip for RT-PCR a set probes, oligonucleotides or cDNAfragments, are selected and optimized from target sequences based on thecriteria of specificity of the hybridization with the target sequence,uniform melting temperature of the probes, and secondary structurestability. The following strategies for efficient selection of probesequences satisfying these criteria are considered: 1) Removal ofexactly repetitive sequences: to efficiently find sequences appearing inmore than one open reading frame (ORF), and remove them from candidatesfor probe sequences. 2) Minimization of frequency of occurrence: toestimate the frequency of occurrence of a probe sequence based on thefrequency of occurrence of all k-tuples consisting of a probe sequence.Only rarely occurring sequences are selected for probe candidates. 3)Unifying melting temperature: to calculate the melting temperature ofmatching probe. The filter classifies probe sequences into fewest groupswith a uniform melting temperature. All DNA probes on a chip surface aresubjected to the same hybridization and wash conditions. Therefore, theuniform melting temperature minimizes false-positive and/orfalse-negative signals, making accurate target identification possible.4) Filtering secondary structure stability: to calculate the free energyof optimal secondary structure of probe. Stable intra-strand secondarystructure of probe hinders rapid hybridization to a target sequence,resulting in extensive decrease of the signal intensity. This filterremoves those unfavorable probe sequences. 5) Minimization of Hammingdistance: to calculate the minimum Hamming distance between a probesequence and a target sequence. It examines the specificity of probe.

On-chip PCR

As shown in FIG. 13, in contrast to conventional PCR, the reaction isperformed directly on chip and reaction products are measured bycapacitance change. The underlying principle of sequence detection isbased on the well-known process of semi-nested PCR. On-chip PCR includesliquid phase PCR with two sequence-specific primers, which is performedon the chip with covalently bound nested, allele-specific PCR primers.PCR products generated in the liquid phase are then re-amplified in asemi-nested PCR directly on the chip surface. Thus in on-chip PCRamplification and sequence detection are combined within a single step.The oligonucleotide microarray is designed such that positive signalsreveal the presence and nature of the target DNA of interest. On-chipPCR can be employed to identify single nucleotide polymorphisms (SNPs)in human genomic DNA. On-chip PCR can be used for detection andidentification of pathogens. This has the benefits of combiningconventional PCR amplification with microarray technology for thedevelopment of simple and rapid DNA diagnostic systems.

Quality Control Methods for the Manufacture of Polymer Arrays

The combined synthesis and detection capabilities of the chips can beused as a method for gaining unprecedented control and monitoring overthe manufacture of polymer arrays. FIG. 14 depicts how this method canwork. In FIG. 14 a given electrode can be activated as described abovein order to carry out an electrochemical reaction of interest.Periodically during this reaction, the CMOS electronics can bereconfigured to provide a reading of the capacitance changes at thiselectrode. The CMOS would then be changed back to a configuration thatto carry on with the electrochemistry reaction. By changing betweenconfigurations over time, it is possible to provide a real time readoutof capacitance changes that have been pre-calibrated to indicatecritical manufacturing process control issues such, but not limited to,the density to at which polymers are being added on a given electrode,the extent to which the polymerization reaction has proceeded, orwhether degradation/side product reactions are occurring. Once a givencapacitance change has been attained (indicated by the Reaction 1 inFIG. 14), another electrochemical reaction of interest is carried out.As before, the CMOS can be reconfigured in real time to monitor this newreaction until desired endpoints are measured. These reaction/monitoringcycles can be carried out through as many iterations as desired, eachtime insuring that they are all occurring within pre-determinedspecifications (i.e., until the last reaction, the Xth reaction in FIG.14, is completed).

“Quality Control Buffers” (QC buffers in FIG. 14) can also be utilizedto test the integrity of the electrode surface once thefunctionalization reactions described above are completed. These QCbuffer treatments can include placing a test solution over thechip/electrode that could test for such features as the resistance ofthe polymer to delamination or other properties of chemical stability.The QC buffers could also include test reagents that mimic theproperties of materials binding to, or reacting with, polymers on theelectrode surfaces. In any case, the effects of these QC buffers on theelectrode surface would be monitored by configuring the chip's CMOS todetermine any capacitance changes once these QC buffers are applied.Note that these reactions could be carried out immediately after thechip's fabrication as a manufacturing quality control. In addition, QCbuffers could be provided with the chip to enable users to carry outtheir own quality control assessments and thereby insure that the chipremains in the functional order to which they have been designed.

This application discloses several numerical range limitations thatsupport any range within the disclosed numerical ranges even though aprecise range limitation is not stated verbatim in the specificationbecause the embodiments of the invention could be practiced throughoutthe disclosed numerical ranges. Finally, the entire disclosure of thepatents and publications referred in this application, if any, arehereby incorporated herein in entirety by reference.

1. A method of synthesizing and monitoring the synthesis of a polymercomprising: activating an electrode to attach a first polymer base tosecond polymer base on a surface of an electrode; detecting acapacitance change on the electrode surface; and activating theelectrode to attach a third polymer base to the second polymer base. 2.The method of claim 1, further comprising comparing the detectedcapacitance change to a pre-calibrated capacitance change.
 3. The methodof claim 2, wherein the comparison is utilized to detect the density toat which polymers bases are being added on a given electrode, the extentto which a polymerization reaction has proceeded, or whetherdegradation/side product reactions are occurring.
 4. The method of claim1, wherein an integrating charge amplifier is utilized to detectcapacitance change on the electrode surface.
 5. The method of claim 1,wherein the polymer is a DNA oligonucleotide, a peptide, or an aptamer.6. The method of claim 1, wherein activating comprises generating avoltage on the electrode.
 7. The method of claim 1, wherein activatingcomprises generating a voltage on the electrode that creates an acidicor basic field that deprotects a polymer base so that it binds on theelectrode surface.
 8. The method of claim 1, wherein the polymer basesare in an aqueous solution or conductive gel.
 9. The method of claim 1,wherein the electrode is part of an array of electrodes fabricated on asingle substrate. 10.-18. (canceled)
 19. A method of quality controlchecking a polymer synthesis or polymer detection device comprising:applying a test solution to a device comprising an electrode; detectinga capacitance change on the device; and comparing the detectedcapacitance change with an expected capacitance change.
 20. The methodof claim 19, wherein the test solution comprises reagents that mimic theproperties of materials binding to, or reacting with, polymers on theelectrode surfaces.
 21. The method of claim 19, wherein the comparisonis utilized to detect the density to at which polymers bases are beingadded on a given electrode, the extent to which a polymerizationreaction has proceeded, or whether degradation/side product reactionsare occurring.
 22. The method of claim 19, wherein an integrating chargeamplifier is connected to the electrode and is configured to detectcapacitance change on the surface of the electrode.
 23. The method ofclaim 19, wherein the test solution comprises an aqueous solution orconductive gel.
 24. The method of claim 19, wherein the electrode ispart of an array of electrodes fabricated on a single substrate. 25.(canceled)
 26. The method of claim 1, wherein the capacitance change isdetected and the electrode is activated using a same circuitry.
 27. Amethod comprising: deprotecting a protected polymer base flowed over anelectrode by applying a voltage to the electrode using a circuitry;detecting a capacitance change on a surface of the electrode using thesame circuitry wherein the capacitance change is caused by attachment ofthe deprotected polymer base to the electrode.
 28. The method of claim27, wherein the protected polymer base is a known polymer base.
 29. Themethod of claim 27, wherein the circuitry comprises an integratingcharge amplifier.
 30. The method of claim 27, wherein the deprotectedpolymer base is attached to a polymer pre-attached to the electrode. 31.The method of claim 30, further comprising determining a sequence of thepolymer pre-attached to the electrode.